Cellular mediators of innate immunity do not express TcR, yet have the capacity to recognize and respond to target cells in the absence of antibody. In this project we have used redirected lysis to demonstrate that a number of adhesion molecules, including CD38, CD44, CD56, and CD69, have the ability to trigger cytotoxic responses in human NK cells and neutrophils. These responses however are dependent upon cytokines, and represent events secondary to the direct recognition of pathogenic substances. Recently an ancient family of pathogen recognition receptors, the toll like receptors (TLRs) was discovered in human and mouse EST data bases, and two of these, TLR2 and 4 were cloned and shown to trigger inflammatory responses to LPS. We have begun examining TLR expression in human cells, and have found easily detectible message of several of the TLRs in monocytes, dendritic cells, NK cells, T cells and PMN. Monocytes express the highest levels of TLR message, and these levels decrease during differentiation of monocytes to dendritic cells. A mAb to TLR1 stains all monocytes strongly, but gives lower level staining on dentritic cells. Co-transfection of TLR1 with TLR2 in 293 cells potentiates the response of TLR2 to some forms of LPS, but inhibits the response in other systems. Our first priority is to determine the expression patterns of TLRs and then to study TLR signaling in immunocytes and in cells from peripheral tissues. Toward this end we have cloned TLR1 and 5, and are in the process of cloning (or obtaining from others) the other TLRs. Our intention is to raise antibodies to each of the TLRs, and to use them to study protein expression, and to do crosslinking studies. The immune response to a pathogen begins by its recognition by the innate system, resulting in the development of an inflammatory response. It is likely that the TLRs play a major role in this process.Redirected cytotoxicity has been used in preclinical mouse models to block tumor growth, but at late stages tumor growth frequently results in profound immunosuppression of both T and B cell responses, thus reducing the effectiveness of any immunotherapeutic approach. We originally showed that tumor growth also resulted in a selective loss in STAT5 protein expression in B and T cell compartments, which could lead to unresponsiveness to several cytokines including IL-2. To further our understanding of the immune suppression, we have, in collaboration with Dr. Enzo Bronte, used an in vitro system to study immunosuppression. Brontes group originally found that immunosuppression was induced by a suppressor type macrophage/dendritic cell that invades the lymphoid tissue at later stages of tumor growth, and he has immortalized these cells. We have found that these cells strongly block T cell proliferation in response to stimulation by antigen, allogeneic cells and Con A. Moreover, inhibition of proliferation is due to the induction of a non- responsiveness to IL-2. Earlier events, such as up regulation of CD69 and CD25, and induction of IL-2 secretion are not impaired. The non- responsive state is dependent on nitric oxide production. We have shown that IFN- , produced by splenocytes, induces the production of nitric oxide in the suppressor cells, and that antibody to IFN- and an inhibitor of iNOS (inducible nitric oxide synthase) both block the induction of the non-responsive state. Non-responsiveness is not accompanied by a loss in STAT5 (A and B) expression-that occurs later, but there is a marked reduction tyrosine phosphorylation of STAT5. Early results have not yet detected a change in JAK3 expression or phosphorylation. These results suggest that the induction of iNOS in macrophage-related cells plays a pivotal role in determining whether the cells will stimulate or suppress immune responses, and we intend to study iNOS and its product in macrophages that have been subjected to various differentiation signals. - Adhesion Molecules, bispecific antibodies, Cellular cytotoxicity, Immunosuppression, redirected cytolysis, signaling molecules, Innate Immunity, - Human Tissues, Fluids, Cells, etc. & Neither Human Subjects nor Human Tissues